Nrs 2008 Valaitis 001.Pdf
Total Page：16
File Type：pdf, Size：1020Kb
ARTICLE IN PRESS Insect Biochemistry and Molecular Biology 38 (2008) 611– 618 Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb Bacillus thuringiensis pore-forming toxins trigger massive shedding of GPI-anchored aminopeptidase N from gypsy moth midgut epithelial cells Algimantas P. Valaitis à USDA Forest Service, 359 Main Road, Delaware, OH 43015, USA article info abstract Article history: The insecticidal Cry proteins produced by Bacillus thuringiensis strains are pore-forming toxins (PFTs) Received 16 November 2007 that bind to the midgut brush border membrane and cause extensive damage to the midgut epithelial Received in revised form cells of susceptible insect larvae. Force-feeding B. thuringiensis PFTs to Lymantria dispar larvae elicited 3 March 2008 rapid and massive shedding of a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase N (APN) Accepted 9 March 2008 from midgut epithelial cells into the luminal fluid, and depletion of the membrane-anchored enzyme on the midgut epithelial cells. The amount of APN released into the luminal fluid of intoxicated larvae was Keywords: dose- and time-dependent, and directly related to insecticidal potency of the PFTs. The induction of Bacillus thuringiensis toxin-induced shedding of APN was inhibited by cyclic AMP and MAPK kinase (MEK) inhibitors Pore-forming toxins PD98059 and U0126, indicating that signal transduction in the MEK/ERK pathway is involved in the Aminopeptidase N Shedding regulation of the shedding process. APN released from epithelial cells appears to be generated by the Signal transduction action of a phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of the GPI anchor based upon detection of a cross-reacting determinant (CRD) on the protein shed into the luminal fluid. Alkaline phosphatase was also released from the gut epithelial cells, supporting the conclusion that other GPI-anchored proteins are released as a consequence of the activation PI-PLC. These observations are the basis of a novel and highly sensitive tool for evaluating the insecticidal activity of new Cry proteins obtained though discovery or protein engineering. Published by Elsevier Ltd. 1. Introduction 2001). In other insects, APNs, cadherins, a GPI-anchored alkaline phosphatase, and lipids have been characterized as toxin-binding Bacillus thuringiensis (Bt) is an aerobic, gram-positive bacter- receptors for various Cry proteins (Vadlamudi et al., 1995; Jurat- ium that is used as a biopesticide for the control of insect pests. Fuentes and Adang, 2004; Griffitts et al., 2005). Most Bt strains produce parasporal proteinaceous crystals con- Until recently, it was generally accepted that the toxic effect of taining one or more pore-forming insecticidal proteins referred to the Cry proteins was due to osmotic lysis of the cells as a result of as Cry toxins (Schnepf et al., 1998). The insecticidal activity of Cry the permeabilization of the epithelial membranes. However, proteins has been attributed to their ability to form ionic pores in bacterial pore-forming toxins can promote cell death that is not the brush border membrane of midgut epithelial cells after directly related to membrane permeabilization. For example, the interaction with specific receptors (Li et al., 1991). Several unique activity of Pasteurella multicida toxin (PMT) appears to be directly receptors for Bt toxins that may mediate the restricted host related to activation of a phosphatidylinositol-specific phospho- specificity of Cry toxins towards target insect pests have been lipase, initiating a cascade of signaling events and a wide identified in different insect species. A GPI-anchored aminopepti- spectrum of cellular responses (Seo et al., 2000). Cry toxins have dase (APN-1) and a 270 kDa glycoconjugate (BTR-270) have been been reported to induce activation of adenylate cyclase and the identified as high-affinity binding proteins for Cry toxins in elevation of intracellular cyclic AMP in intoxicated insect cell Lymantria dispar larvae. APN-1 was found to exhibit unique cultures. However, whether cyclic AMP plays a pivotal role in the specificity in its interaction with only Cry1Ac. In contrast, BTR-270 pathogenic process in the activity of Bt remains unclear (Knowles displayed high affinity interaction with a diverse number of highly and Farndale, 1998; Zhang et al., 2006). toxic Cry toxins, suggesting that it is a common receptor for the A large number of membrane-associated proteins, including insecticidal proteins (Valaitis et al., 1997, 2000; Valaitis et al., cytokines, growth factors and their receptors, cell adhesion molecules, and syndecans are secreted as biologically active molecules into the extracellular environment by a process à Tel.: +1740 368 0020; fax: +1740 369 2601. referred to as shedding. Most shedding processes have been E-mail address: [email protected] found to be mediated by a proteolytic cleavage (Almquist and 0965-1748/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.ibmb.2008.03.003 ARTICLE IN PRESS 612 A.P. Valaitis / Insect Biochemistry and Molecular Biology 38 (2008) 611–618 Carlsson, 1988). The specific proteases involved in the shedding PMSF. Purification of the Cry1Ac toxin-binding APN-1 from gypsy processes have been called sheddases or secretases and have been moth BBMV and generation of a polyclonal antibody specific identified as members of the hydroxamate-sensitive ADAM family towards Ld APN-1 was described previously (Valaitis et al., 1995). of membrane-associated proteases (Blobel, 2004). Although BBMV from control and Cry1Ac-intoxicated insect larvae were release of soluble forms of GPI-anchored proteins can be incubated with 0.2 U/ml of phosphatidylinositol-specific phos- generated by endogenous membrane-associated phospholipases, pholipase (PI-PLC) purchased from Sigma for 1 h at 37 1C. The PI- ADAMs have also been implicated in the shedding of some GPI- PLC digests were centrifuged at 14,000g for 20 min and the anchored molecules (Wilhelm et al., 1999; Cavallone et al., 2001; amount of APN released was measured spectrophotometrically Elwood et al., 1991). The bacterial PFTs, streptolysin O (SLO) and using leucine p-nitroanilide as a substrate as described below. The hemolysin (HlyA), induce the shedding of receptors for interleukin soluble aminopeptidase N (sAPN) in the luminal fluid from Cry1A 6 and lipopolysaccharide, respectively (Walev et al., 1996), and toxin-treated insect larvae was purified by chromatography using Staphylococcus aureus a-toxin and the anthrax cholesterol binding a 5 ml Macroprep High Q ion-exchange column and concentrated pore-forming factor (AnlO) activate shedding of syndecans (Park by ultra filtration using YM-30 ultra filtration membrane for et al., 2004; Popova et al., 2006). The kinetics of the shedding immunochemical analysis. processes induced by bacterial PFTs is rapid, paralleling the kinetics of pore formation. One common feature of these shedding 2.3. Bacterial strains and purification of toxins processes is that the activation is dependent on intracellular signaling pathways which can be blocked with specific inhibitors Bacillus thuringiensis subsp. kurstaki (HD-1 and HD-73), subsp. of the mitogen-activated protein kinase (MAPK) extracellular tolworthi (HD125), subsp. sotto, subsp. tenebrionis, a crystal minus regulated kinase (ERK) pathway (Park et al., 2004). In addition, derivative of subsp. sotto (4E3 Cry-) and Escherichia coli strains activation of the p38 MAPK signaling pathway has been demon- expressing the cloned Cry1Aa, Cry1Ab, Cry1Ac, Cry2A and Cry3A strated to play a role in cellular defense and recovery after attack toxins were obtained from the Bacillus Genetic Stock Center at The by PFTs (Husmann et al., 2006; Aroian and van der Goot, 2007). Ohio State University. Sporulated cultures were produced by A specific aminopeptidase N isozyme (APN-1) has been growing the Bt stains on nutrient agar plates for 6–7 days and identified as toxin-binding receptor in L. dispar, based on ligand from 0.5 L cultures of Bt strains grown at 30 1C for 5–6 days in a blotting and surface plasmon resonance binding studies (Jenkins medium containing 1% glucose, 0.2% peptone, 0.5% NZ amine-A et al., 2000). However, subsequent efforts to demonstrate a casein hydrolysate, 0.2% yeast extract, 15 mM (NH ) SO ,23mM functional role for APN-1 in L. dispar, as well as other APNs in 4 2 4 KH PO ,27mMKHPO , 1 mM MgSO , 0.6 mM CaCl ,17mM insects, in the mode of action of Bt, have been only partially 2 4 2 4 4 2 ZnSO ,17mM CuSO and 2 mM FeSO . The Cry1A proteins in HD- successful (Garner et al., 1999; Gill and Ellar, 2002; Sivakumar 4 4 4 1, HD-73 and Bt sotto preparations were extracted from washed et al., 2007; Pigott and Ellar, 2007) The major finding of this study spore/crystal pellets obtained from sporulated Bt cultures by is that Bt Cry proteins are shown to induce massive shedding of solubilization of the Cry proteins using 0.1 M carbonate, 10 mM APN-1 into the luminal fluid mediated by activation of GPI- EDTA and 10 mM DTT at pH 10.4 for 30 min at room temperature. specific phospholipase C via an intracellular signaling pathway. The solubilized Cry protoxins were activated by digestion with Since shedding of various cell surface proteins induced by affinity-purified gypsy moth trypsin (Valaitis, 1995) for 30 min at bacterial pathogens has been implicated in promoting their room temperature, and the activated toxins were purified by gel virulence, shedding of APN may promote the cytocidal action of filtration using a 3.5  46 cm column of Sephacryl S-200 equili- Bt Cry toxins. brated in pH 9.6 50 mM carbonate–bicarbonate buffer containing 0.2 M NaCl and 10 mM EDTA. Cry2A was obtained from carbonate- extracted HD-1 spore/crystal pellets by solubilization of the 2. Materials and methods crystals in 0.1 M NaOH and subsequently purified using Sephacryl S-200 gel filtration.